Methods and compositions for treatment of neurological disorder

ABSTRACT

The present invention provides methods and compositions for producing a neurosalutary effect in a subject useful in treatment of neurological disorders, including retinal and optic nerve damage, in a subject in need thereof. The method includes administering to a subject a therapeutically effective amount of a hexose, such as mannose.

BACKGROUND OF THE INVENTION

Nerves in mature mammals, such as the optic nerve, do not normallyregenerate after injury. Retinal ganglion cells (RGCs) initiate asprouting reaction at their damaged nerve endings, but this growth isabortive and the cells soon begin to die (Ramon y Cajal, 1991).Nonetheless, RGCs can regenerate lengthy axons through a peripheralnerve graft (Aguayo et al., 1991) and even through the optic nerveitself if the lens is injured (Fischer et al., 2000; Leon et al., 2000),or if a fragment of peripheral nerve is implanted into the vitreous(Berry et al., 1996). These latter manipulations lead to the appearanceof activated macrophages in the eye, and it has been recently shown thatintravitreal macrophage activation is sufficient to allow RGCs toregenerate their axons through the optic nerve (Leon et al., 2000; Yinet al., 2003). In culture, a macrophage-derived protein, acting inconcert with a small molecule that is constitutively present in thevitreous, stimulates mature rat RGCs to regenerate their axons in acAMP-dependent fashion (Yin et al., 2003).

In contrast to mammals, fish and amphibia can regenerate their opticnerves throughout life (Jacobson, 1991). In culture, the most potentaxon-promoting factor for goldfish RGCs is a small hydrophilic molecule(<500 Daltons) that is secreted by non-neuronal cells of the opticnerve. This molecule is referred to as AF-1 (Schwalb et al., 1995;Schwalb et al., 1996).

Understanding the factors involved in mammalian and non-mammalian neuronregeneration will aid in the development of potential therapeutics fortreatment of neuronal disorders. Disorders of the peripheral and centralnervous system are widespread, and for many of these conditionseffective therapeutic interventions are lacking. Neurological disordersmay be caused by an injury to a neuron, such as a mechanical injury oran injury due to a toxic compound, by the abnormal growth or developmentof a neuron, or by the misregulation (such as downregulation orupregulation) of an activity of a neuron. There is a need in the art formethods and compositions that can improve the ability of a neuron, orportion of the nervous system, to resist insult, to regenerate, and tomaintain desirable function, which can be used for treatment ofneurological disorders.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for producing aneurosalutary effect in a subject with a neurological condition; sucheffects include promoting neuronal survival, axonal outgrowth, neuronalregeneration or normalized neurological function in a subject.

In one aspect, the present invention provides a method which includesadministering to a subject a therapeutically effective amount of ahexose (e.g., mannose) or a hexose derivative, thereby producing aneurosalutary effect in the subject.

In another embodiment, the present invention provides treatment ofneurological disorders, including retinal and optic nerve damage, in asubject by administering to a subject in need of such treatment aneffective amount of a hexose or a hexose derivative.

In other embodiments, the methods of the invention further includeadministering to a subject a cAMP modulator and/or a macrophage-derivedfactor.

In one aspect, the hexose or hexose derivative is administered to asubject in accordance with the present invention such that the hexose isbrought into contact with neurons of the central nervous system of thesubject. For example, the hexose may be administered into thecerebrospinal fluid of the subject into the intrathecal space byintroducing the hexose into a cerebral ventricle, the lumbar area, orthe cistema magna. In such circumstances, the hexose can be administeredlocally to cortical neurons or retinal ganglion cells to produce aneurosalutary effect.

In certain embodiments, the pharmaceutically acceptable formulationprovides sustained delivery, providing effective amounts of the hexoseto a subject for at least one week; or in other embodiments, at leastone month, after the pharmaceutically acceptable formulation isinitially administered to the subject. Approaches for achievingsustained delivery of a formulation of the invention include the use ofa slow release polymeric capsule, a bioerodible matrix, or an infusionpump that disperses the hexose or other therapeutic compound of theinvention. The infusion pump may be implanted subcutaneously,intracranially, or in other locations as would be medically desirable.In certain embodiments, the therapeutic factors or compositions of theinvention would be dispensed by the infusion pump via a catheter eitherinto the cerebrospinal fluid, or to a site where local delivery wasdesired, such as a site of neuronal injury or a site ofneurodegenerative changes.

Pharmaceutical compositions that include a hexose derivative and apharmaceutically acceptable carrier may be packed with instructions foruse of the pharmaceutical composition for treating a neurologicaldisorder. In one embodiment, the pharmaceutical composition furtherincludes a cAMP modulator and/or a macrophage-derived factor.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-B show axon-promoting effects of a low molecular weight factorfrom the rat vitreous humor. FIG. 1A shows molecules present in eitherthe normal rat vitreous or in the vitreous one week after lens injurywere extracted into saline; molecules<3 kDa in size were separated byultrafiltration and tested for axon-promoting activity using goldfishretinal ganglion cells. When present at either 1.25% or 5% of theiroriginal concentration, the low molecular weight extract induced as muchoutgrowth as inosine, and this was independent of whether or not thelens was injured. Outgrowth data are normalized by subtracting the levelof growth in culture media alone and then dividing by the net growth inthe positive controls. FIG. 1B illustrates cell survival. The number ofretinal ganglion cells per 200× microscope field was unaltered by any ofthe manipulations.

FIG. 2 shows properties of the low molecular weight vitreous-derivedfactor.

Axon growth induced by the low molecular weight factor from the vitreousextract (VE<3) is inhibited completely by the purine analog6-thioguanine (6-TG) but is not affected by an inhibitor of purinetransport, NBTI. These agents exert similar effects on outgrowth inducedby AF-1, a small factor derived from goldfish optic nerve conditionedmedia. In contrast, growth stimulated by inosine is only partiallyinhibited by 6-TG and is blocked completely by NBTI.

FIGS. 3A-C show that the vitreous-derived factor stimulates axon growthin rat retinal ganglion cells in a cAMP-dependent fashion. FIG. 3A showsaxon outgrowth. By itself, the low molecular weight factor from thevitreous has a small but significant axon-promoting effect on ratretinal ganglion cells in culture. The addition of either forskolin(forsk) or the PKA agonist Sp-cAMP-s strongly potentiates this effect.This growth is strongly inhibited by 6-TG. Results are normalized to thelevel of growth seen in control cultures grown in media alone (in therange of 8-12% of cells extending axons>2 cell diameters in length).FIG. 3B shows cell survival. None of the agents tested altered RGCsurvival. **p<0.01 compared to negative control; ^([***])p<0.001compared to growth in cultures treated with PKA agonists alone;^([††])p<0.01 compared to growth in cultures treated with VE<3 plus thePKA agonists. FIG. 3C shows VE<3 gives a near-maximal response at aconcentration of 5%. **p<0.01 compared to negative control;^([***])p<0.001 compared to either negative controls or cells treatedwith PKA agonists alone.

FIG. 4 shows axon growth in goldfish RGCs is not enhanced by elevatingintracellular cAMP levels. Neither forskolin nor Sp-cAMP-s by itselfstimulated axon growth in goldfish RGCs, and reduced the level ofoutgrowth induced by AF-1. The PKA antagonists Rp-cAMP-s or H-89 (at 5μM) did not reduce AF-1 induced growth, although higher concentrationsof H89 (20 μM) did. **p<0.01 (significance of reduction compared togrowth with AF-1 alone).

FIGS. 5A-F illustrate isolation of the small vitreous-derived growthfactor. FIGS. 5A and B show reversed-phase HPLC. The low molecularweight factor from bovine vitreous was concentrated, extracted into 95%ethanol, and subjected to HPLC on a C-18 reversed-phase column. The axonpromoting activity eluted in the earliest peak. FIGS. 5C and D show gelfiltration chromatography. On a G-10 Sephadex column, the axon-promotingactivity eluted as a coherent peak that included high levels of materialshowing adsorbance at 214 nm.

FIGS. 5E and F show normal-phase chromatography. The peak containing theaxon-promoting activity from the gel-filtration column was separated ona LC-NH₂ normal-phase column using HPLC. The axon-promoting activityeluted later than most components with absorbance at 214 nm (arrows).Bioassays in all cases were carried out on goldfish retinal ganglioncells.

FIGS. 6A-E show identification of the axon-promoting factor by massspectrometry. FIGS. 6A and B show fast atomic bombardment mass spectrain the negative ion mode in the range of m/z=140 to 220 of a fractionfrom the LC-NH₂ column that does not induce axon growth (6A) and theadjacent fraction that stimulates axon growth (6B). Spectra were carriedout in the presence of glycerol. Only the active fraction contains apeak with m/z=179.2 (arrow in b). Because the ion masses in the negativemode represent the masses minus one proton, the actual mass of thespecies present in the active fraction would be expected to be 180.FIGS. 6C and D show fast atomic bombardment mass spectra in the positiveion mode in the range of m/z=230 to 300 of a fraction from the LC-NH₂column that does not induce axon growth (6C) and the adjacent fractionthat stimulates axon growth (6D). Spectra were carried out in thepresence of glycerol. Only the active fraction contains peaks withm/z=273.12 and 255.13 (arrows). Because the ion masses in the positivemode represent the masses plus one proton, and because these ions mayinclude adducts with glycerol (mass=92), the actual masses of thespecies present in the active fraction could be 180.13 and 162.12. FIG.6E shows mass spectrum of the m/z=273 species from (6D) subjected toMS/MS analysis in the positive ion mode in the presence of glycerol.When subjected to higher voltage, the m/z 273 species generated aglycerol peak (m/z=93, red asterisk) and an ion of m/z 181 (doublearrows), i.e., the parent species minus glycerol; most of the additionalions represent successive losses of 18 amu from either the 181 ion(m/z=163, 145 and 127, arrows) or from the glycerol adduct of the 181species (m/z=255, 237, arrows). These results indicate that theaxon-promoting factor is a hexose sugar with the formula C₆H₁₂O₆.

FIGS. 7A-F show that goldfish RGCs regenerate their axons in response toD-glucose and D-mannose. FIG. 7A shows outgrowth in response tomonosaccharides. Of the many hexose sugars tested, only glucose (glc)and mannose (man) induced axon growth. Other monosaccharides testedinclude myo-inositol (myo-inos), fructose (fruc), sorbose (sorb),altrose (alt), allose (all), gulose (gul), galactose (gal) and talose(tal), all at 50 or 100 μM and all in the D-configuration. FIG. 7B showscell survival. The effects of mannose and glucose on axon regenerationare not related to any changes in cell survival. FIG. 7C shows adose-response curve for mannose. The effect of mannose on axon outgrowthsaturates at 25-50 μM, and the ED₅₀ is approximately 10 μM. FIG. 7Dshows the effect of mannose is enhanced by a factor which itself has noaxon-promoting effects. By itself, D-glc stimulates only about 70% thelevel of axon growth seen with the vitreous extract. A fraction thatelutes later from the gel-filtration column (14-17) has no activity byitself, but enhances the effect of glucose back to the level of theunfractionated vitreous extract.

FIG. 7E shows that the membrane-permeable cAMP analog, dBcAMP (1 mM)does not augment the effect of glucose on goldfish retinal ganglioncells. FIG. 7F shows that the protein kinase A inhibitor KT5720 haslittle effect on mannose-induced growth in goldfish retinal ganglioncells.

FIGS. 8A-E show effects of various glucose analogs and inhibitors onaxon growth. FIG. 8A shows the stereospecificity of the effects seenwith D-mannose and D-glucose as demonstrated by the inactivity of theL-enantiomers (100 μM). D-glucosamine (25 μM), an glucose analog with anamino group substituted for the hydroxyl group at C-2, is stronglyactive, whereas mannosamine is not. No activity is seen with2-deoxy-D-glucose (1 mM), 3-O-methyl-D-glucose, methyl-α- ormethyl-β-glucose pyranose, N-acetyl-glucosamine (N-Ac-glucosamine) orL-fucose. However, the non-membrane-permeable 6-phosphates of glucoseand mannose (D-glc-6-P, D-man-6-P) induce a small but statisticallysignificant level of outgrowth. **p<0.01 relative to control cells grownin media alone; ***p<0.001 relative to negative controls. FIG. 8B showsthat forskolin diminishes the effect of D-glucose. Outgrowth induced by10 μM glucose is inhibited by forskolin (10 μM). ***p<0.001 compared togrowth induced by glucose alone. FIG. 8C shows 3-O-MG, a non-metabolizedglucose analog and inhibitor of glucose transport, fails to block theeffect of glucose (10 μM). Another inhibitor of glucose transport, 2-DG,does block the effect of glucose, but this effect may be nonspecific,because it also blocks the effect of inosine. FIG. 8D shows thatmannoheptulose (MH, 10 mM), an inhibitor of both glucose-6 kinase andhexose-6 kinase, has no effect on outgrowth induced by glucose ormannose, despite being detrimental to cell survival as shown in FIG. 8Ewhere MH decreases cell survival (c/f, viable RGCs per field).

FIG. 8F shows that extracellular D-glucose-6-phosphate andD-mannose-6-phosphate stimulate a modest amount of outgrowth at 100 μM(p<0.01), and appreciable growth at 1 mM. The y-axis is the same as in(b). **p<0.01, ***p<0.001 relative to cells grown in media alone:^(††)p<0.01 (decrease in survival relative to glucose alone).

FIGS. 9A-E show rat retinal ganglion cells respond selectively tomannose in a cAMP-dependent manner. FIG. 9A shows axon outgrowth.Mannose induced a small, marginally significant level of outgrowth,whereas forskolin (5 μM) by itself produced a somewhat larger effect. Inthe presence of forskolin, mannose induced a striking increase inoutgrowth; glucose at physiological concentrations (5 mM) did not. Theeffect of mannose was at least as great as that of the low molecularextract from the vitreous, and was not altered by the addition ofglucose. These studies were carried out in the presence of 5% fetalbovine serum in the culture media. ^([*]p)=0.05 compared to negativecontrol, 1-tailed t-test; **p<0.01 compared to negative control,2-tailed t-test. ***p<0.001; n.s.=not significant relative to growthinduced by forskolin alone. FIG. 9B shows cell survival. The effect ofmannose and forskolin on axon outgrowth is independent of any changes incell survival. FIG. 9C shows mature rat RGCs respond strongly to mannosein the presence of forskolin. Glucose is inactive. All sugars are in theD-configuration unless noted otherwise. (*)p<0.05 compared to negativecontrol; *p<0.05, ***p<0.001 compared to forskolin alone (dotted line).FIG. 9D shows cell survival is unaffected by any of the carbohydrates.The numbers of viable RGCs per field are normalized to the value innegative controls. FIG. 9E shows proteins (>3 kDa) secreted by activatedmacrophages enhance the effects of mannose. ***p<0.001 compared to anyof the other conditions.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions for producing aneurosalutary effect in a subject useful in treatment of neurologicaldisorders, including nerve damage. The method includes administering toa subject a therapeutically effective amount of a hexose (e.g., mannose)or a hexose derivative.

As used herein, the term “hexose” includes any hexose, or derivativethereof, that is able to produce a neurosalutary effect. Preferredhexoses include D-mannose and gulose. The term “hexose derivative”refers to a hexose molecule that has one or more residues (e.g. esters,ethers, amino groups, amido groups, phosphate groups, sulphate groups,carboxyl groups, carboxy-alkyl groups, and combinations thereof)covalently or ionically attached to one or more of the moleculeshydroxyl groups and able to produce a neurosalutary effect. A preferredderivative includes glucose-6-phosphate. The term hexose derivativeincludes D- and L-isomers of hexose or hexose derivatives able toproduce a neurosalutary effect in a subject. Hexose derivatives are wellknown in the art and commercially available.

As used herein, a “neurosalutary effect” means a response or resultfavorable to the health or function of a neuron, of a part of thenervous system, or of the nervous system generally. Examples of sucheffects include improvements in the ability of a neuron or portion ofthe nervous system to resist insult, to regenerate, to maintaindesirable function, to grow or to survive. The phrase “producing aneurosalutary effect” includes producing or effecting such a response orimprovement in function or resilience within a component of the nervoussystem. For example, examples of producing a neurosalutary effect wouldinclude stimulating axonal outgrowth after injury to a neuron; renderinga neuron resistant to apoptosis; rendering a neuron resistant to a toxiccompound such as β-amyloid, ammonia, or other neurotoxins; reversingage-related neuronal atrophy or loss of function; or reversingage-related loss of cholinergic innervation.

The term “cAMP modulator” includes any compound which has the ability tomodulate the amount, production, concentration, activity or stability ofcAMP in a cell, or to modulate the pharmacological activity of cellularcAMP. cAMP modulators may act at the level of adenylate cyclase,upstream of adenylate cyclase, or downstream of adenylate cyclase, suchas at the level of cAMP itself, in the signaling pathway that leads tothe production of cAMP. Cyclic AMP modulators may act inside the cell,for example at the level of a G-protein such as Gi, Go, Gq, Gs and Gt,or outside the cell, such as at the level of an extra-cellular receptorsuch as a G-protein coupled receptor. Cyclic AMP modulators includeactivators of adenylate cyclase such as forskolin; nonhydrolyzableanalogues of cAMP including 8-bromo-cAMP, 8-chloro-cAMP, or dibutyrylcAMP (db-cAMP); isoprotenol; vasoactive intestinal peptide; calciumionophores; membrane depolarization; macrophage-derived factors thatstimulate cAMP; agents that stimulate macrophage activation such aszymosan or IFN-γ; phosphodiesterase inhibitors such as pentoxifyllineand theophylline; specific phosphodiesterase IV (PDE IV) inhibitors; andbeta 2-adrenoreceptor agonists such as salbutamol. The term cAMPmodulator also includes compounds which inhibit cAMP production,function, activity or stability, such as phosphodiesterases, such ascyclic nucleotide phosphodiesterase 3B. cAMP modulators which inhibitcAMP production, function, activity or stability are known in the artand are described in, for example, in Nano et al., 2000, the contents ofwhich are incorporated herein by reference.

“Phosphodiesterase IV inhibitor” refers to an agent that inhibits theactivity of the enzyme phosphodiesterase IV. Examples ofphosphodiesterase IV inhibitors are known in the art and include4-arylpyrrolidinones, such as rolipram (A.G. Scientific, Inc.),nitraquazone, denbufylline, tibenelast, CP-80633 and quinazolinedionessuch as CP-77059.

“Beta-2 adrenoreceptor agonist” refers to an agent that stimulates thebeta-2 adrenergic receptor. Examples of beta-2 adrenoreceptor agonistsare known in the art and include salmeterol, fenoterol andisoproterenol.

As used herein, the term “macrophage-derived factor” includes any factorderived from a macrophage that has the ability to produce aneurosalutary effect in a subject. Macrophage-derived factors include,but are not limited to, peptides such as oncomodulin and TGF-β. See, WO01/091783, the disclosure of which is incorporated herein by reference.

The term “administering” to a subject includes dispensing, delivering orapplying an active compound in a pharmaceutical formulation to a subjectby any suitable route for delivery of the active compound to the desiredlocation in the subject, including delivery by either the parenteral ororal route, intramuscular injection, subcutaneous/intradermal injection,intravenous injection, buccal administration, transdermal delivery andadministration by the rectal, colonic, vaginal, intranasal orrespiratory tract route. A hexose may, for example, be administered to acomatose, anesthetized or paralyzed subject via an intravenous injectionor may be administered intravenously to a pregnant subject to produce aneurosalutary effect in the fetus. Specific routes of administration mayinclude topical application (such as by eyedrops, creams or erodibleformulations to be placed under the eyelid), intraocular injection intothe aqueous or the vitreous humor, injection into the external layers ofthe eye, such as via subconjunctival injection or subtenon injection,parenteral administration or via oral routes.

As used herein, the language “contacting” is intended to include both invivo or in vitro methods of bringing a compound of the invention intoproximity with a neuron such that the compound can exert a neurosalutaryeffect on the neuron.

As used herein, the term “effective amount” includes an amounteffective, at dosages and for periods of time necessary, to achieve thedesired result, such as sufficient to produce a neurosalutary effect ina subject. An effective amount of an active compound as defined hereinmay vary according to factors such as the disease state, age, and weightof the subject, and the ability of the active compound to elicit adesired response in the subject. Dosage regimens may be adjusted toprovide the optimum therapeutic response. An effective amount is alsoone in which any toxic or detrimental effects of the active compound areoutweighed by the therapeutically beneficial effects.

A non-limiting range for a therapeutically effective concentration ofactive compound, e.g., mannose, is 5 μM to 1 mM. In a preferredembodiment the therapeutically effective concentration of the activecompound is 25-500 μM.

A therapeutically effective amount or dosage of an active compound mayrange from about 0.001 to 30 mg/kg body weight, with other ranges of theinvention including about 0.01 to 25 mg/kg body weight, about 0.1 to 20mg/kg body weight, about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to7 mg/kg, and 5 to 6 mg/kg body weight. The skilled artisan willappreciate that certain factors may influence the dosage required toeffectively treat a subject, including but not limited to the severityof the disease or disorder, previous treatments, the general healthand/or age of the subject, and other diseases present. Moreover,treatment of a subject with a therapeutically effective amount of anactive compound can include a single treatment or a series oftreatments. In one example, a subject is treated with an active compoundin the range of between about 0.1 to 20 mg/kg body weight, one time perweek for between about 1 to 10 weeks, alternatively between 2 to 8weeks, between about 3 to 7 weeks, or for about 4, 5, or 6 weeks. Itwill also be appreciated that the effective dosage of an active compoundused for treatment may increase or decrease over the course of aparticular treatment.

The term “subject” is intended to include animals. In particularembodiments, the subject is a mammal, a human or nonhuman primate, adog, a cat, a horse, a cow or a rodent.

“Neurological disorder” is intended to include a disease, disorder, orcondition which directly or indirectly affects the normal functioning oranatomy of a subject's nervous system. Elements of the nervous systemsubject to disorders which may be effectively treated with the compoundsand methods of the invention include the central, peripheral, somatic,autonomic, sympathetic and parasympathetic components of the nervoussystem, neurosensory tissues within the eye, ear, nose, mouth or otherorgans, as well as glial tissues associated with neuronal cells andstructures. Neurological disorders may be caused by an injury to aneuron, such as a mechanical injury or an injury due to a toxiccompound, by the abnormal growth or development of a neuron, or by themisregulation (such as downregulation or upregulation) of an activity ofa neuron. Neurological disorders can detrimentally affect nervous systemfunctions such as the sensory function (the ability to sense changeswithin the body and the outside environment); the integrative function(the ability to interpret the changes); and the motor function (theability to respond to the interpretation by initiating an action such asa muscular contraction or glandular secretion). Examples of neurologicaldisorders include traumatic or toxic injuries to peripheral or cranialnerves, spinal cord or to the brain, cranial nerves, traumatic braininjury, stroke, cerebral aneurism, and spinal cord injury. Otherneurological disorders include cognitive and neurodegenerative disorderssuch as Alzheimer's disease, dementias related to Alzheimer's disease(such as Pick's disease), Parkinson's and other Lewy diffuse bodydiseases, senile dementia, Huntington's disease, Gilles de la Tourette'ssyndrome, multiple sclerosis, amyotrophic lateral sclerosis, hereditarymotor and sensory neuropathy (Charcot-Marie-Tooth disease), diabeticneuropathy, progressive supranuclear palsy, epilepsy, andJakob-Creutzfieldt disease. Autonomic function disorders includehypertension and sleep disorders. Also to be treated with compounds andmethods of the invention are neuropsychiatric disorders such asdepression, schizophrenia, schizoaffective disorder, Korsakoff spsychosis, mania, anxiety disorders, or phobic disorders, learning ormemory disorders (such as amnesia and age-related memory loss),attention deficit disorder, dysthymic disorder, major depressivedisorder, mania, obsessive-compulsive disorder, psychoactive substanceuse disorders, anxiety, phobias, panic disorder, bipolar affectivedisorder, psychogenic pain syndromes, and eating disorders. Otherexamples of neurological disorders include injuries to the nervoussystem due to an infectious disease (such as meningitis, high fevers ofvarious etiologies, HIV, syphilis, or post-polio syndrome) and injuriesto the nervous system due to electricity (including contact withelectricity or lightning, and complications from electro-convulsivepsychiatric therapy). The developing brain is a target for neurotoxicityin the developing central nervous system through many stages ofpregnancy as well as during infancy and early childhood, and the methodsof the invention may be utilized in preventing or treating neurologicaldeficits in embryos or fetuses in utero, in premature infants, or inchildren with need of such treatment, including those with neurologicalbirth defects. Further neurological disorders include, for example,those listed in Harrison's Principles of Internal Medicine (Braunwald etal., McGraw-Hill, 2001) and in the American Psychiatric Association'sDiagnostic and Statistical Manual of Mental Disorders DSM-IV (AmericanPsychiatric Press, 2000) both incorporated herein by reference in theirentirety. Neurological disorders associated with ophthalmic conditionsinclude retina and optic nerve damage, glaucoma and age related maculardegeneration.

The term “stroke” is art recognized and is intended to include suddendiminution or loss of consciousness, sensation, and voluntary motioncaused by rupture or obstruction (for example, by a blood clot) of anartery of the brain.

“Traumatic brain injury” is art recognized and is intended to includethe condition in which, a traumatic blow to the head causes damage tothe brain or connecting spinal cord, often without penetrating theskull. Usually, the initial trauma can result in expanding hematoma,subarachnoid hemorrhage, cerebral edema, raised intracranial pressure,and cerebral hypoxia, which can, in turn, lead to severe secondaryevents due to low cerebral blood flow.

The term “outgrowth” includes the process by which axons or dendritesextend from a neuron. The outgrowth can result in a new neuriticprojection or in the extension of a previously existing cellularprocess. Axonal outgrowth may include linear extension of an axonalprocess by 5 cell diameters or more. Neuronal growth processes,including neuritogenesis, can be evidenced by GAP-43 expression detectedby methods such as immunostaining. “Modulating axonal outgrowth” meansstimulating or inhibiting axonal outgrowth to produce salutatory effectson a targeted neurological disorder.

The term “CNS neurons” is intended to include the neurons of the brain,the cranial nerves and the spinal cord.

Various aspects of the invention are described in further detail in thefollowing subsections:

Pharmaceutically Acceptable Formulations

Pharmaceutical compositions and packaged formulations comprising ahexose derivative and a pharmaceutically acceptable carrier are alsoprovided by the invention. These pharmaceutical compositions may alsoinclude a macrophage-derived factor and/or a cAMP modulator.

In a method of the invention, the hexose derivative (e.g., mannose),optionally in conjunction with a macrophage-derived factor and/or a cAMPmodulator, can be administered in a pharmaceutically acceptableformulation. Such pharmaceutically acceptable formulation may includethe hexose derivative as well as a pharmaceutically acceptablecarrier(s) and/or excipient(s). As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and anti fungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible. Forexample, the carrier can be suitable for injection into thecerebrospinal fluid. Excipients include pharmaceutically acceptablestabilizers and disintegants. The present invention pertains to anypharmaceutically acceptable formulations, including synthetic or naturalpolymers in the form of macromolecular complexes, nanocapsules,microspheres, or beads, and lipid-based formulations includingoil-in-water emulsions, micelles, mixed micelles, synthetic membranevesicles, and resealed erythrocytes.

In one embodiment, the pharmaceutically acceptable formulations comprisea polymeric matrix. The terms “polymer” or “polymeric” areart-recognized and include a structural framework comprised of repeatingmonomer units which is capable of delivering a hexose derivative suchthat treatment of a targeted condition, such as a neurological disorder,occurs. The terms also include co-polymers and homopolymers such assynthetic or naturally occurring. Linear polymers, branched polymers,and cross-linked polymers are also meant to be included.

For example, polymeric materials suitable for forming thepharmaceutically acceptable formulation employed in the presentinvention, include naturally derived polymers such as albumin, alginate,cellulose derivatives, collagen, fibrin, gelatin, and polysaccharides,as well as synthetic polymers such as polyesters (PLA, PLGA),polyethylene glycol, poloxomers, polyaphydrides, and pluronics. Thesepolymers are biocompatible with the nervous system, including thecentral nervous system, they are biodegradable within the centralnervous system without producing any toxic byproducts of degradation,and they possess the ability to modify the manner and duration of theactive compound release by manipulating the polymer's kineticcharacteristics. As used herein, the term “biodegradable” means that thepolymer will degrade over time by the action of enzymes, by hydrolyticaction and/or by other similar mechanisms in the body of the subject. Asused herein, the term “biocompatible” means that the polymer iscompatible with a living tissue or a living organism by not being toxicor injurious and by not causing an immunological rejection. Polymers canbe prepared using methods known in the art.

The polymeric formulations can be formed by dispersion of the activecompound within liquefied polymer, as described in U.S. Pat. No.4,883,666, the teachings of which are incorporated herein by referenceor by such methods as bulk polymerization, interfacial polymerization,solution polymerization and ring polymerization as described in OdianG., Principles of Polymerization and ring opening polymerization, 2nded., John Wiley & Sons, New York, 1981, the contents of which areincorporated herein by reference. The properties and characteristics ofthe formulations are controlled by varying such parameters as thereaction temperature, concentrations of polymer and the active compound,the types of solvent used, and reaction times.

The active therapeutic compound can be encapsulated in one or morepharmaceutically acceptable polymers, to form a microcapsule,microsphere, or microparticle, terms used herein interchangeably.Microcapsules, microspheres, and microparticles are conventionallyfree-flowing powders consisting of spherical particles of 2 millimetersor less in diameter, usually 500 microns or less in diameter. Particlesless than 1 micron are conventionally referred to as nanocapsules,nanoparticles or nanospheres. For the most part, the difference betweena microcapsule and a nanocapsule, a microsphere and a nanosphere, ormicroparticle and nanoparticle is size; generally there is little, ifany, difference between the internal structure of the two. In one aspectof the present invention, the mean average diameter is less than about45 μm, preferably less than 20 μm, and more preferably between about 0.1and 10 μm.

In another embodiment, the pharmaceutically acceptable formulationscomprise lipid-based formulations. Any of the known lipid-based drugdelivery systems can be used in the practice of the invention. Forinstance, multivesicular liposomes, multilamellar liposomes andunilamellar liposomes can all be used so long as a sustained releaserate of the encapsulated active compound can be established. Methods ofmaking controlled release multivesicular liposome drug delivery systemsare described in PCT Application Publication Nos: WO 9703652, WO9513796, and WO 9423697, the contents of which are incorporated hereinby reference.

The composition of the synthetic membrane vesicle is usually acombination of phospholipids, usually in combination with steroids,especially cholesterol. Other phospholipids or other lipids may also beused.

Examples of lipids useful in synthetic membrane vesicle productioninclude phosphatidylglycerols, phosphatidylcholines,phosphatidylserines, phosphatidylethanolamines, sphingolipids,cerebrosides, and gangliosides, with preferable embodiments includingegg phosphatidylcholine, dipalmitoylphosphatidylcholine,distearoylphosphatidyleholine, dioleoylphosphatidylcholine,dipalmitoylphosphatidylglycerol, and dioleoylphosphatidylglycerol.

In preparing lipid-based vesicles containing an active compound suchvariables as the efficiency of active compound encapsulation, labilityof the active compound, homogeneity and size of the resulting populationof vesicles, active compound-to-lipid ratio, permeability, instabilityof the preparation, and pharmaceutical acceptability of the formulationshould be considered.

Prior to introduction, the formulations can be sterilized, by any of theumerous available techniques of the art, such as with gamma radiation orelectron beam sterilization.

Ophthalmic products for topical use may be packaged in multidose form.Preservatives are thus required to prevent microbial contaminationduring use. Suitable preservatives include: benzalkonium chloride,thimerosal, chlorobutanol, methyl paraben, propyl paraben, phenylethylalcohol, edetate disodium, sorbic acid, polyquaternium-1, or otheragents known to those skilled in the art. Such preservatives aretypically employed at a level of from 0.001 to 1.0% weight/volume (“1%w/v”). Such preparations may be packaged in dropper bottles or tubessuitable for safe administration to the eye, along with instructions foruse.

Administration of the Pharmaceutically Acceptable Formulation

The pharmaceutically acceptable formulations of the invention areadministered such that the active compound comes into contact with asubject's nervous system to thereby produce a neurosalutary effect. Bothlocal and systemic administration of the formulations are contemplatedby the invention. Desirable features of local administration includeachieving effective local concentrations of the active compound as wellas avoiding adverse side effects from systemic administration of theactive compound. In one embodiment, the active compound is administeredby introduction into the cerebrospinal fluid of the subject. In certainaspects of the invention, the active compound is introduced into acerebral ventricle, the lumbar area, or the cistema magna. In anotheraspect, the active compound is introduced locally, such as into the siteof nerve or cord injury, into a site of pain or neural degeneration, orintraocularly to contact neuroretinal cells.

The pharmaceutically acceptable formulations can be suspended in aqueousvehicles and introduced through conventional hypodermic needles or usinginfusion pumps.

In one embodiment, the active compound formulation described herein isadministered to the subject in the period from the time of, for example,an injury to the CNS up to about 100 hours after the injury hasoccurred, for example within 24, 12, or 6 hours from the time of injury.

In another embodiment of the invention, the active compound formulationis administered into a subject intrathecally. As used herein, the term“intrathecal administration” is intended to include delivering an activecompound formulation directly into the cerebrospinal fluid of a subject,by techniques including lateral cerebroventricular injection through aburrhole or cistemal or lumbar puncture or the like (described inLazorthes et al., 1991, and Ommaya A. K., 1984, the contents of whichare incorporated herein by reference). The term “lumbar region” isintended to include the area between the third and fourth lumbar (lowerback) vertebrae. The term “cistema magna” is intended to include thearea where the skull ends and the spinal cord begins at the back of thehead. The ten-n “cerebral ventricle” is intended to include the cavitiesin the brain that are continuous with the central canal of the spinalcord. Administration of an active compound to any of the above mentionedsites can be achieved by direct injection of the active compoundformulation or by the use of infusion pumps. Implantable or externalpumps and catheter may be used.

For injection, the active compound formulation of the invention can beformulated in liquid solutions, preferably in physiologically compatiblebuffers such as Hank's solution or Ringer's solution. In addition, theactive compound formulation may be formulated in solid form andre-dissolved or suspended immediately prior to use. Lyophilized formsare also included. The injection can be, for example, in the form of abolus injection or continuous infusion (such as using infusion pumps) ofthe active compound formulation.

In one embodiment of the invention, the active compound formulation isadministered by lateral cerebroventricular injection into the brain of asubject, preferably within 100 hours of when an injury (resulting in acondition characterized by aberrant axonal outgrowth of central nervoussystem neurons) occurs (such as within 6, 12, or 24 hours of the time ofthe injury). The injection can be made, for example, through a burr holemade in the subject's skull. In another embodiment, the formulation isadministered through a surgically inserted shunt into the cerebralventricle of a subject, preferably within 100 hours of when an injuryoccurs (such as within 6, 12 or 24 hours of the time of the injury). Forexample, the injection can be made into the lateral ventricles, whichare larger, even though injection into the third and fourth smallerventricles can also be made. In yet another embodiment, the activecompound formulation is administered by injection into the cistemamagna, or lumbar area of a subject, preferably within 100 hours of whenan injury occurs (such as within 6, 12, or 24 hours of the time of theinjury).

An additional means of administration to intracranial tissue involvesapplication of compounds of the invention to the olfactory epithelium,with subsequent transmission to the olfactory bulb and transport to moreproximal portions of the brain. Such administration can be by nebulizedor aerosolized prerparations.

In another embodiment of the invention, the active compound formulationis administered to a subject at the site of injury, preferably within100 hours of when an injury occurs (such as within 6, 12, or 24 hours ofthe time of the injury).

In a further embodiment, ophthalmic compositions of the presentinvention are used to prevent or reduce damage to retinal and opticnerve head tissues, as well as to enhance functional recovery afterdamage to ocular tissues. Ophthalmic conditions that may be treatedinclude, but are not limited to, retinopathies (including diabeticretinopathy and retrolental fibroplasia), macular degeneration, ocularischemia, glaucoma. Other conditions to be treated with the methods ofthe invention include damage associated with injuries to ophthalmictissues, such as ischemia reperfusion injuries, photochemical injuries,and injuries associated with ocular surgery, particularly injuries tothe retina or optic nerve head by exposure to light or surgicalinstruments. The ophthalmic compositions may also be used as an adjunctto ophthalmic surgery, such as by vitreal or subconjunctival injectionfollowing ophthalmic surgery. The compounds may be used for acutetreatment of temporary conditions, or may be administered chronically,especially in the case of degenerative disease. The ophthalmiccompositions may also be used prophylactically, especially prior toocular surgery or noninvasive ophthalmic procedures or other types ofsurgery.

Duration and Levels of Administration

In a preferred embodiment of the method of the invention, the activecompound is administered to a subject for an extended period of time toproduce a neurosalutary effect, such as effect modulation of axonaloutgrowth. Sustained contact with the active compound can be achievedby, for example, repeated administration of the active compound over aperiod of time, such as one week, several weeks, one month or longer.More preferably, the pharmaceutically acceptable formulation used toadminister the active compound provides sustained delivery, such as“slow release” of the active compound to a subject. For example, theformulation may deliver the active compound for at least one, two,three, or four weeks after the pharmaceutically acceptable formulationis administered to the subject. Preferably, a subject to be treated inaccordance with the present invention is treated with the activecompound for at least 30 days (either by repeated administration or byuse of a sustained delivery system, or both).

As used herein, the term “sustained delivery” is intended to includecontinual delivery of the active compound in vivo over a period of timefollowing administration, preferably at least several days, a week,several weeks, one month or longer. Sustained delivery of the activecompound can be demonstrated by, for example, the continued therapeuticeffect of the active compound over time (such as sustained delivery ofthe macrophage-derived factor can be demonstrated by continuedproduction of a neurosalutary effect in a subject). Alternatively,sustained delivery of the active compound may be demonstrated bydetecting the presence of the active compound in vivo over time.

Preferred approaches for sustained delivery include use of a polymericcapsule, a minipump to deliver the formulation, a bloerodible implant,or implanted transgenic autologous cells (as described in U.S. Pat. No.6,214,622). Implantable infusion pump systems (such as Infusaid; seesuch as Zierski, J. et al, 1988; Kanoff, R. B., 1994) and osmotic pumps(sold by Alza Corporation) are available in the art. Another mode ofadministration is via an implantable, externally programmable infusionpump. Suitable infusion pump systems and reservoir systems are alsodescribed in U.S. Pat. No. 5,368,562 by Blomquist and U.S. Pat. No.4,731,058 by Doan, developed by Pharmacia Deltec Inc.

It is to be noted that dosage values may vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of the activecompound and that dosage ranges set forth herein are exemplary only andare not intended to limit the scope or practice of the claimedinvention.

The invention, in another embodiment, provides a pharmaceuticalcomposition consisting essentially of a hexose derivative and apharmaceutically acceptable carrier, as well as methods of use thereofto modulate axonal outgrowth by contacting CNS neurons with thecomposition. By the term “consisting essentially of” is meant that thepharmaceutical composition does not contain any other modulators ofneuronal growth such as, for example, nerve growth factor (NGF). In oneembodiment, the pharmaceutical composition of the invention can beprovided as a packaged formulation. The packaged formulation may includea pharmaceutical composition of the invention in a container and printedinstructions for administration of the composition for producing aneurosalutary effect in a subject having a neurological disorder. Use ofa hexose derivative in the manufacture of a medicament for modulatingthe axonal outgrowth of neurons is also encompassed by the invention.

In Vitro Treatment of CNS Neurons

Neurons derived from the central or peripheral nervous system can becontacted with a hexose derivative in vitro to modulate axonal outgrowthin vitro. Accordingly, neurons can be isolated from a subject and grownin vitro, using techniques well known in the art, and then treated inaccordance with the present invention to modulate axonal outgrowth.Briefly, a neuronal culture can be obtained by allowing neurons tomigrate out of fragments of neural tissue adhering to a suitablesubstrate (such as a culture dish) or by disaggregating the tissue, suchas mechanically or enzymatically, to produce a suspension of neurons.For example, the enzymes trypsin, collagenase, elastase, hyaluronidase,DNase, pronase, dispase, or various combinations thereof can be used.Methods for isolating neuronal tissue and the disaggregation of tissueto obtain isolated cells are described in Freshney, Culture of AnimalCells, A Manual of Basic Technique, Third Ed., 1994, the contents ofwhich are incorporated herein by reference.

Such cells can be subsequently contacted with a hexose (alone or incombination with a macrophage-derived factor and/or a cAMP modulator) inamounts and for a duration of time as described above. Once modulationof axonal outgrowth has been achieved in the neurons, these cells can bere-administered to the subject, such as by implantation.

Screening Assays

The ability of a hexose to produce a neurosalutary effect in a subjectmay be assessed using any of a variety of known procedures and assays.For example, the ability of a hexose derivative to re-establish neuralconnectivity and/or function after an injury, such as a CNS injury, maybe determined histologically (either by slicing neuronal tissue andlooking at neuronal branching, or by showing cytoplasmic transport ofdyes). The ability of compounds of the invention to re-establish neuralconnectivity and/or function after an injury, such as a CNS injury, mayalso be assessed by monitoring the ability of the hexose derivative tofully or partially restore the electroretinogram after damage to theneural retina or optic nerve; or to fully or partially restore apupillary response to light in the damaged eye.

Other tests that may be used to determine the ability of a hexose toproduce a neurosalutary effect in a subject include standard tests ofneurological function in human subjects or in animal models of spinalinjury (such as standard reflex testing, urologic tests, urodynamictesting, tests for deep and superficial pain appreciation, propnoceptiveplacing of the hind limbs, ambulation, and evoked potential testing). Inaddition, nerve impulse conduction can be measured in a subject, such asby measuring conduct action potentials, as an indication of theproduction of a neurosalutary effect.

Animal models suitable for use in the assays of the present inventioninclude the rat model of partial transaction (described in Weidner etal., 2001). This animal model tests how well a compound can enhance thesurvival and sprouting of the intact remaining fragment of an almostfully-transected cord. Accordingly, after administration of the hexosethese animals may be evaluated for recovery of a certain function, suchas how well the rats may manipulate food pellets with their forearms (towhich the relevant cord had been cut 97%).

Another animal model suitable for use in the assays of the presentinvention includes the rat model of stroke (described in Kawamata etal., 1997). This paper describes in detail various tests that may beused to assess sensorimotor function in the limbs as well asvestibulomotor function after an injury. Administration to these animalsof the compounds of the invention can be used to assess whether a givencompound, route of administration, or dosage provides a neurosalutaryeffect, such as increasing the level of function, or increasing the rateof regaining function or the degree of retention of function in the testanimals.

Standard neurological evaluations used to assess progress in humanpatients after a stroke may also be used to evaluate the ability of ahexose to produce a neurosalutary effect in a subject. Such standardneurological evaluations are routine in the medical arts, and aredescribed in, for example, “Guide to Clinical Neurobiology” Edited byMohr and Gautier (Churchill Livingstone Inc. 1995).

For assessing function of the peripheral nervous system, standard testsinclude electromyography, nerve conduction velocity measurements,potentials assessment and upper/lower extremity somato-sensory evokedpotentials. Electromyography tests record the electrical activity inmuscles, and is used to assess the function of the nerves and muscles.The electrode is inserted into a muscle to record its electricalactivity. It records activity during the insertion, while the muscle isat rest, and while the muscle contracts. The nerve conduction velocitytest evaluates the health of the peripheral nerve by recording how fastan electrical impulse travels through it. A peripheral nerve transmitsinformation between the spinal cord and the muscles. A number of nervoussystem diseases may reduce the speed of this impulse. Electrodes placedon the skin detect and record the electrical signal after the impulsetravels along the nerve. A second stimulating electrode is sends a smallelectrical charge along the nerve; the time between the stimulation andresponse will be recorded to determine how quickly and thoroughly theimpulse is sent.

Standard tests for function of the cranial nerves, as known to thoseskilled in the neurological medical art, include facial nerve conductionstudies; orbicularis oculi reflex studies (blink reflex studies);trigeminal-facial nerve reflex evaluation as used in focal facial nervelesions, Bell's palsy, trigeminal neuralgia and atypical facial pain;evoked potentials assessment; visual, brainstem and auditory evokedpotential measurements; thermo-diagnostic small fiber testing; andcomputer-assisted qualitative sensory testing.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting. The contents of allreferences, patents and published patent applications cited throughoutthis application are hereby incorporated by reference.

EXAMPLES Example I Retinal Ganglion Cells Extend Axons in Response toTreatment with Hexose

Goldfish retinal ganglion cells are prepared as described in PCTapplication serial no. PCT/US98/03001, the contents of which areincorporated herein by reference, and contacted with a hexose derivativesuch as mannose. The ability of the hexose derivative to stimulateaxonal outgrowth from the goldfish retinal ganglion cells is monitoredas described in PCT application serial no. PCT/US98/03001.

Example II

Methods

Extraction low molecular weight factor from rat vitreous. Moleculespresent in the rat vitreous were extracted into normal saline (8vitreous bodies in 1.5 ml saline, overnight with mixing, 4° C.). Thevitreous bodies were derived from either normal adult male Fisher rats(Charles River Laboratories, Wilmington, Mass.), 200-250 g, or from rats7 days after lens injury, a time at which we see clear evidence ofGAP-43 upregulation in RGCs after lens injury (Leon et al., 2000). Lensinjury was accomplished using a 30 G needle as described (Leon et al.,2000). The vitreous extract was passed through a 22 μM low-proteinbinding filter (Pall Life Sciences, Ann Arbor, Mich.) to remove cellulardebris; low molecular components were separated by ultrafiltrationthrough a 3 kDa molecular weight cut-off (MWCO) membrane.

Bovine vitreous fluid was extracted from the eyes of newborn calves in aratio of 4 volumes normal saline to 1 volume of vitreous fluid.Extraction took place at 4° C. As above, extracts were passed through a22 μM low protein binding filter (Pall), then a 3 kDa cut-offultrafiltration device to remove higher molecular weight components. Todetermine whether a small molecule from the bovine vitreous behaves likegoldfish AF-1 or inosine in promoting growth, we tested its activity inthe presence or absence of 6-thioguanine (6-TG, 20 μM, Sigma), anantagonist of a purine-sensitive kinase that is important for axongrowth or 4-(nitrobenzyl-6-thioinosine) (NBTI, 20 μM, AldrichChemicals), an inhibitor or purine transport across the cell membrane.

Fractionation of conditioned media. To concentrate the active factor(s)and remove inorganic salts, the low molecular weight extract of bovinevitreous was lyophilized and extracted with 95% ethanol (16% of theoriginal sample volume, 4° C., with frequent Vortexing, 1 hr). Theethanol-soluble fraction was lyophilized, redissolved in 400 μL water,and applied to a C18 reversed-phase HPLC column (Delta Pak 5μ C 18 100Å, Waters, Milford, Mass.: 0.5 ml/min). Buffer A was 0.1%trifluoroacetic acid (TFA) in water, whereas buffer B was 0.08% TFA in amixture of isopropanol, acetonitrile and water in a ratio of 3:2:2. Thegradient for elution was 0% B for 2 minutes and then 0-100% B in 42minutes. Bioassays were performed on pools of fractions representing 2min.

Further separation was achieved by gel-filtration chromatography using aSephadex G-10 (Sigma) column 1.6 cm×48 cm. This column separatesmolecules under 700 Daltons. The sample was concentrated 20×, insolublecomponents were removed by centrifigation, and the soluble portion,which was found to contain all of the biological activity (not shown),was applied in a volume of 0.5 ml. The column was run under pressure at0.3 ml/min using a peristaltic pump. Fractions of 4.5 ml were collectedand tested in the bioassay at a concentration of approximately 5%relative to the original concentration in the sample.

The final stage of purification was achieved using a normal-phase HPLCcolumn (Shodex aminopropyl, Thomson Instruments, Oceanside, Calif.). Thefraction from the G-10 column containing axon-promoting activity waslyophilized and redissolved in 200 μl 80% acetonitrile. Afterpre-equilibrating the column with 75% acetonitrile, the sample wasapplied at a flow rate of 1 ml/min. Fractions of 1 ml were collected andbioassayed.

Goldfish retinal ganglion cell cultures. Mammalian AF-1 was initiallycharacterized in bioassays using dissociated goldfish retinal ganglioncells (RGCs). In brief, Comet variety goldfish (Mt. Parnell Fisheries,PA) were dark-adapted, cooled to 4° C., and killed by cervicaltransection. Retinas were dissected and dissociated by incubation inpapain followed by a series of trituration steps that result in anRGC-enriched cell suspension (Schwartz & Agronoff,; Schwalb et al.,1995). Approximately 500 RGCs per well were plated together with theexperimental samples to be tested in defined, serum-free mediacontaining Leibovitz' L15 media (Gibco BRL) plus N1 and N2 supplements(Bottenstein, Saito), antioxidants, and bovine serum albumin asdescribed in detail elsewhere (Schwalb et al., 1995). In eachexperiment, 4 wells of each sample were distributed in 24-well plates ina blinded fashion so that the person evaluating axon outgrowth wasunaware of the type of sample present in each well. Experiments includeda positive control (previously validated goldfish AF-1 or inosine) and anegative control (defined media alone), likewise distributed on theplate in quadruplicate in a blinded fashion. After 6 days in culture at21° C., axon outgrowth, operationally defined as the percent of RGCsthat extended an axon≧5 cell diameters in length, was evaluated inapproximately 150 consecutively encountered RGCs per well. Data wereanalyzed by averaging axon growth in the 4 wells for each sample,subtracting the level of growth found in negative controls (typically3-5%), and normalizing by the net growth in positive controls (usually20-40%). Data are presented as normalized means±SEM. All experimentswere repeated at least 3 times.

Rat retinal ganglion cell cultures. Retinal ganglion cells wereidentified by retrograde labeling with Fluorogold (FG: Fluorochrome,Inc.). For this, adult male Sprague-Dawley rats were anesthetized,placed in a stereotaxic apparatus, and the superior colliculi wereexposed by removing the overlying posterior cerebral cortex. FG wasinjected into several sites in the superior colliculi bilaterally. Inaddition, a small piece of Gelfoam (Upjohn, Kalamazoo, Mich.)impregnated with FG was placed over the superior colliculi, the scalpwound closed, and the skin sutured closed. After allowing 7 days for FGto be retrogradely transported up the optic nerve, rats were killed,eyes were removed, and the retinas dissected. Retinas were dissociatedwith papain treatment followed by trituration. Mixed cultures containing100-150 FG-labeled RGCs per well were maintained in 24 well plates in adefined, serum-free media (Minimal Essential Media containing NaHCO₃,)at 37° C. at 5% CO₂ for 3 days. As with the goldfish cultures, allexperimental samples were tested in quadruplicate and were distributedacross the culture dishes in a randomized fashion. Statistical handlingof the data was as described above.

Mass spectrometry. FAB mass spectra were obtained at the Michigan StateUniversity Mass Spectrometry Facility using a JEOL. HX-110double-focusing mass spectrometer (JEOL USA, Peabody, Mass.) operatingin the positive or negative ion mode. Ions were produced by bombardmentwith a beam of Xe atoms (6 keV). The accelerating voltage was 10 kV andthe resolution was set at 3000. For FAB-CAD-MS/MS, helium was used s thecollision gas in a cell located in the first field-free region. Thehelium pressure was adjusted to reduce the abundance of the parent ionby 50%. A data system generated linked scans at a constant ratio ofmagnetic to electrical fields (B/E). The instrument was scanned from m/z50 to 2000. Spectra presented were from a single scan.

Results

A small axon-promoting factor is constitutively present in the mammalianvitreous. Lens injury stimulates retinal ganglion cells to regenerateinjured axons through the optic nerve in vivo (Leon et al., 2000). Toinvestigate the factors that might stimulate this growth, we extractedthe molecules that are present in the normal vitreous body, or in thevitreous body one week after nerve crush and lens injury, into saline.Molecules smaller than 3 kDa were separated by ultrafiltration andtested for axon-promoting activity using goldfish retinal ganglion cellsas a bioassay (Schwalb et al., 1995; Benowitz et al., 1998). The lowmolecular weight extract showed full activity when diluted 80-fold,regardless of whether or not the lens had been injured (FIG. 1). Atone-tenth this concentration, it was ineffective (data not shown).

The small axon-promoting factor is present in bovine vitreous andbehaves like goldfish AF-1. To isolate sufficient quantities of thesmall growth factor and analyze its structure, we investigated whetherit was present in the bovine vitreous. A low molecular weight componentof the bovine vitreous extract (VE<3), when tested at an 80-folddilution, induced as much outgrowth from goldfish RGCs asgoldfish-derived AF-1 (FIG. 2). To determine whether thevitreous-derived factor behaves like AF-1, we examined whether itsactivity could be blocked by either of two agents: 6-thioguanine, whichnon-competitively blocks the effect of AF-1 on outgrowth but which iscompetitive with inosine (Petrausch et al., 2000); or NBTI, an inhibitorof purine transport across the cell membrane, which blocks the effectsof inosine but does not diminish the activity of AF-1 (Benowitz et al.,1998). NBTI had very little effect on growth induced by VE<3 or AF-1,but effectively blocked growth induced by inosine. In contrast, 6-TGbrought growth induced by VE<3 or AF-1 below baseline levels, but onlypartially blocked the effects of inosine (FIG. 2). Thus, the lowmolecular weight factor from the vitreous behaves like goldfish AF-1.

The small vitreous-derived factor stimulates axon regeneration in adultrat RGCs in a cAMP-dependent manner. Retinas of adult rats weredissociated and cultured as described in Materials and Methods; RGCswere identified by virtue of retrograde labeling with Fluorogold priorto preparing the cultures. In the absence of additional factors,approximately 5%-8% of RGCs extended axons>2 cell diameters in lengthafter 3 days. Elevating intracellular cAMP with forskolin or with themembrane permeable, nonhydrolyzable cAMP analog Sp-cAMPs had littleeffect on outgrowth. The low molecular weight extract from the vitreousstimulated a small but significant level of growth on its own (p<0.01),and the combination of this small factor with either forskolin orSp-cAMPs greatly potentiated this effect (FIG. 3 a: p<0.001 comparinggrowth with the low molecular weight factor plus either forskolin orSp-cAMPs with the growth obtained with any one alone). As shown abovefor goldfish ROCs, 6-TG reduced growth induced by the low-molecularweight factor plus forskolin (p<0.01) or by the low molecular weightfactor plus Sp-cAMPs (p<0.05). Thus, the low molecular weight factorfrom the vitreous stimulates rat RGCs to extend axons in acAMP-dependent fashion; like axon growth in goldfish RGCs, outgrowth ismediated through a purine-sensitive mechanism. The effects of the lowmolecular weight fragment were unrelated to any changes in cell survival(FIG. 3 b). Maximal effects were attained even when VE<3 was diluted to4% of its original concentration in the vitreous (FIG. 3 c).

Unlike rat RGCs, goldfish RGCs showed a robust response to VE<3 evenwithout increasing intracellular [cAMP]_(i) (FIG. 4). Neither forskolin(10 μM) nor Sp-cAMPs (150 μM) induced growth on its own, in conformitywith previous results showing that membrane-permeable analogs of cAMP orcGMP do not stimulate goldfish RGCs to extend axons (Benowitz, 1998).When added to AF-1, forskolin and Sp-cAMPs caused a non-statisticallysignificant decrease in outgrowth. Conversely, growth induced by AF-1was not diminished by the membrane-permeable, nonhydrolyzable PKAantagonist Rp-cAMPs (100 μM) nor the PKA inhibitor H89 (5 μM). At 20 μM,H89 diminished the effect of AF-1 (p<0.001), although at thisconcentration H-89 affects other kinases besides PKA. Thus, the responseof goldfish RGCs to the low molecular weight factor is considerably lesscAMP-dependent than the response of rat RGCs.

Isolation of the active component. The low molecular weight fraction ofthe vitreous extract was concentrated by lyophilization and extractedinto a small volume of 95% ethanol. This resulted in nearly completerecovery of the axon-promoting activity while removing approximately 98%of the inorganic salts (data not shown). Following separation on areversed-phase C18 column, the axon-promoting activity was not retainedby the column and eluted as part of a large absorbance peak in the firstfew minutes (FIG. 5 a,b).

Further purification was achieved by gel-filtration chromatography on aSephadex G-10 column. The axon-promoting activity was largely containedwithin a single fraction (165-180 min) that overlapped with a majorabsorbance peak (FIG. 5 c, 5 d). Even at high concentrations, thisfraction induced only 60-80% the level of axon-promoting activity as thestarting material. Material that elutes later on the G-10 column andwhich has no activity by itself, when added back to the active fraction,brings its activity back to the level of the starting material (data notshown). When the active fraction from the G-10 column was applied to anormal-phase column (LC-NH₂), the activity eluted late, in a region thatshowed very little absorbance (FIG. 5 e, 5 f). Thus, normal-phasechromatography resulted in a high degree of purification. The activefraction again stimulated about 80% as much outgrowth as the positivecontrol (starting material). This fraction was concentrated and runagain on the same column, collecting fractions coinciding withabsorbance peaks visualized with the detector set at the highest levelof UV sensitivity. Bioassay revealed that the axon-promoting activitywas concentrated in a single peak eluting at 9.8 min (data not shown).

Identification of the active factor by mass spectrometry. The purifiedfractions containing the axon-promoting activity and adjacent inactivepeaks were analyzed in the positive and negative ion modes by Fast AtomBombardment (FAB) mass spectrometry. Principal results were obtained inthe presence of glycerol and were confirmed in the presence oftetraethylammonium and nitrobenzoic acid. In the negative ion mode, theactive fraction (#17) contained an ion with m/z=179.2 (arrow) that wasabsent in the adjacent inactive fraction (#16) (FIGS. 6 a, b). Becausem/z values in the negative ion mode correspond to the true mass minus 1proton, the molecule present in the active fraction is predicted to havea mass of 180. In the positive ion mode, two peaks appeared in thebiologically active fraction that were absent in the adjacent inactivefraction (m/z=273.2 and 255.2: FIGS. 6 c, d, arrows). Because m/z valuesin the positive ion mode contain an additional proton, the two uniqueions are predicted to have masses of 272.2 and 254.2; however, if theseions represent glycerol adducts (mass=92) of the parent species, themass of the larger one would be 180, which is similar to that found inthe negative ion mode, while the other would be 162. Further analysis ofthe m/z=273 ion by MS/MS in the positive ion mode (FIG. 6 e) confirmedthe presence of glycerol (m/z=93, asterisk) and the 180 mass (m/z=181,double arrows). MS/MS also generated peaks corresponding to the parentspecies minus multiples of 18, i.e., 163, 145, and 127 (arrows). Thelatter peaks are likely to represent serial losses of hydroxyl groupsfrom the 181 ion, whereas the peaks with m/z=255 and 237 probablyrepresent glycerol adducts of the 163 and 145 ions, respectively. Theseresults predict that the active molecule is a carbohydrate with theformula C₆H₁₂O₆ (mass=180).

Specific carbohydrates induce axon regeneration from goldfish RGCs Basedupon the mass spectrometry results, we tested the axon-promoting effectsof hexose sugars and related compounds on RGCs in culture. In goldfishRGCs, myo-inositol, the ketoses fructose and sorbose, and the aldosesD-allose, D-altrose, D-gulose, D-talose, and D-galactose all failed tostimulate outgrowth (all tested at 50 or 100 μM: FIG. 7 a). However, thetwo structurally related aldose sugars mannose and glucose stimulated asmuch outgrowth as the low molecular weight factor isolated from thebovine vitreous extract via gel-exclusion and normal-phasechromatography (c.f. FIG. 5 d, f). The L-enantiomers of glucose andmannose were inactive (data not shown). Outgrowth in response to mannosesaturated at 25-50 μM, and the ED₅₀ was approximately 10 μM (FIG. 7 c).A similar dose-response curve was obtained for glucose (data not shown).As mentioned above, the growth-promoting factor derived from bovinevitreous retained only 60-80% of the original activity after isolationby size-exclusion and normal-phase chromatography (FIG. 5 d,f), and fullactivity could be restored by adding back a later fraction from thesize-exclusion column which itself did not cause any growth. Similarly,adding the same late-eluting fraction from the size-exclusion column to50 μM mannose or glucose increased activity back to the level of thevitreous extract prior to fractionation (FIG. 7 d). The effect ofglucose (FIG. 7 e) or mannose (not shown) on goldfish RGCs was notenhanced with the membrane-permeable, non-hydrolyzable cAMP analogdBcAMP. Conversely, the protein kinase A inhibitor KT5720, at thenormally effective dosage of 1 μM, did not diminish the effect ofmannose, and had only a slight effect at 10 μM (FIG. 7 f). Likewise, thePKA inhibitor Rp-cAMPs had no effect on glucose-induced outgrowth at thenormally effective dose of 100 μM, but blocked growth by 48% when testedat 1 mM (not shown).

The culture media used in these experiments already provides highconcentrations of galactose (5 mM) and pyruvate (5 mM) for energymetabolism and as a carbon source. Thus, the outgrowth induced by lowmicromolar concentrations of mannose or glucose is unlikely to berelated to energy metabolism. This is further suggested by the fact thatadding 5 mM methyl pyruvate, which is readily used for ATP synthesis toour media, had no effect (data not shown). Further evidence that theeffects of glucose and mannose on axon growth are unrelated to energymetabolism comes from the fact that low micromolar concentrations ofthese carbohydrates have no effect on cell survival (FIG. 7 b).

To examine the specificity of the observed effects and to gain insightsinto possible structure-function relationships, we examined thebiological activity of several related compounds. The L-enantiomers ofglucose and mannose were completely inactive (FIG. 8 a). D-glucosaminewas strongly active (p<0.001); D-mannosamine was ineffective, as were2-deoxy-D-glucose, 3-O-methyl-D-glucose, methyl-α-D-glucopyranoside,methyl-β-D-glucopyranoside, acetylglucosamine and fucose. D-glucose- andD-mannose-6-phosphate stimulated a relatively modest but statisticallysignificant amount of outgrowth (p<0.01). Because these latter twophosphate derivatives carry a negative charge, they do not pass throughthe cell membrane. This would suggest that glucose and mannose maystimulate axon outgrowth via an extracellular mechanism. Further supportfor this possibility comes from studies using the inhibitor of glucosetransport, 3-O-methyl glucose (3-O-MG). In a molar ratio of 100:1,3-O-MG failed to block the effects of glucose (FIG. 8 b). We alsoinvestigated the effects of another glucose transport inhibitor,2-deoxyglucose (2-DG). However, at concentrations as low as 1 mM, 2-DGhad nonspecific effects on axon growth, and blocked the effects of bothglucose and inosine; inosine has been shown to stimulate axon growth viaan intracellular mechanism (Benowitz et al., 1998. As shown above, theeffect of AF-1 on goldfish RGCs is not enhanced by elevatingintracellular cAMP levels using either forskolin or Sp-cAMP-s, andactually appears to decline (FIG. 4). Likewise, the effect of glucose ongoldfish RGCs is diminished by the addition of forskolin (FIG. 8 c).

In several cell types, glucose is sensed by the activity of hexokinases,the first enzymes in the glycolytic pathway, or by the concentration ofdownstream metabolites (Rolland et al., 2001). In goldfish RGCs,mannoheptulose (MH, 10 mM), an inhibitor of both glucose-6 kinase andhexose-6 kinase, had no effect on outgrowth induced by glucose ormannose, despite being detrimental to cell survival (FIGS. 8 d and 8 e).Thus, the glucose/mannose sensor for axon growth is not the kinaseinvolved in the first step of glycolysis, nor does it depend on theintracellular concentration of the 6-phosphate derivatives or of anydownstream metabolites. The failure of MH to block outgrowth even in theface of diminished cell survival provides further evidence that theaxon-promoting effect of D-glucose or D-mannose is unrelated to cellsurvival.

When introduced extracellularly, D-glucose-6-phosphate andD-mannose-6-phosphate stimulated a modest amount of outgrowth at 100 μM(p<0.01), and appreciable growth at 1 mM (p<0.001) (FIG. 8 f). The6-phosphate derivatives are negatively charged and do not pass throughthe cell membrane (Abeles et al., 1992). This suggests that the6-phosphate derivatives, and by extension glucose and mannosethemselves, may stimulate axon outgrowth via an extracellular sensor.

Rat RGCs respond selectively to mannose in a cAMP-dependent manner. Likegoldfish RGCs, RGCs from the mature rat also grow axons in response tomicromolar concentrations of monosaccharides, but with severalinteresting differences. Rat RGCs show a marginally significant responseto either mannose (p=0.05, one-tailed) or VE<3 alone (p=0.05,two-tailed), and a small but significant response to forskolin alone(FIG. 9 a). In the presence of forskolin, mannose in micromolarconcentrations more than doubled the level axon growth induced byforskolin alone (p<0.001). Mannose-induced growth was at least as highas that induced by low molecular weight components of unfractionatedbovine vitreous extract, and was not augmented further by the additionof high concentrations of glucose. In other studies, mannose was seen toachieve near-maximal effects at 50 μM (data not shown). In contrast,glucose, even at millimolar concentrations as occur in vivo and in thepresence of forskolin failed to induce growth above the level offorskolin alone. FIG. 9 b shows the effect is not related to cellsurvival. Thus, whereas goldfish RGCs extend lengthy axons in responseto either glucose or mannose without augmenting intracellular cAMP, ratRGCs respond selectively to mannose in a cAMP-dependent manner.

RGCs from the mature rat showed a far greater response selectivity thangoldfish RGCs. D-mannose by itself had little effect on rat RGCs (FIG. 9a and FIG. 9 c), but in the presence of forskolin, it increased axonoutgrowth 3-fold over baseline (p<0.001: FIGS. 9 a and 9 c). This effectwas similar to that of VE<3 (c.f. FIG. 3), and was likewise unrelated tochanges in cell survival (FIG. 9 b and FIG. 9 d). In the presence offorskolin, mannose elicited maximal effects by 50 μM, with an ED₅₀ ofapproximately 10 μM (data not shown). Stereospecificity is demonstratedby the inactivity of L-mannose (FIG. 9 c). Under similar conditions,glucose had no effect whatsoever (FIG. 9 c), even when present atmillimolar concentrations (FIG. 9 a). Of the other sugars tested, gulosehad some activity (p<0.05) (FIG. 9 c). Unlike goldfish RGCs, rat RGCsdid not respond to glucosamine (FIG. 9 c) or to high concentrations ofmannose-6-phosphate (up to 10 mM: data not shown).

Additive effect of a macrophage-derived factor (s) and mannose in ratRGCs. In culture, a 10-20 kDa macrophage-derived protein potentiates theresponse of rat RGCs to a small vitreous-derived growth factor when[cAMP]i is elevated (Yin et al., 2003). In the presence of forskolin,macrophage-conditioned media nearly doubled the response of rat RGCs tomannose, elevating growth almost 6-fold above baseline (FIG. 9 e). Thisgrowth corresponds to over 50% of cultured RGCs extending axons in 3days.

Discussion

We show here that specific monosaccharides stimulate retinal ganglioncells to regenerate their axons, and that this effect is independent ofenergy metabolism. Even when other energy sources are abundant, goldfishRGCs respond to low micromolar concentrations of either glucose ormannose to regenerate their axons. Responsiveness to these carbohydratesdoes not require elevation of intracellular cAMP. Rat RGCs, on the otherhand, are more selective. Rat RGCs extend axons in response tomicromolar concentrations of mannose in a cAMP-dependent fashion, butnot to glucose at levels similar to those found in vivo. The response ofrat RGCs can be augmented considerably further by macromoleculessecreted by macrophages. These findings may help explain in part thedifferent regenerative capacities of lower and higher vertebrates invivo. In the goldfish, the abundance of glucose may suffice to enableRGCs to regenerate their axons after injury. In the rat, however,although mannose is abundant in the vitreous humor, its' role instimulating axon regeneration is likely to be permissive rather than aregulatory. By itself, mannose is insufficient to induce axonregeneration in vivo or in vitro. However, in the presence of elevatedintracellular cAMP, mannose potentiates the effects ofmacrophage-derived factors, and hence is likely to play a role in thedramatic axon regeneration that is seen after macrophage activation invivo (Yin et al., submitted).

In cell culture, media conditioned by the non-neuronal cells of thegoldfish optic nerve was found to stimulate extensive axon regenerationfrom RGCs. Partial purification revealed that most of this activitycould be attributed to a small, hydrophilic molecule that was termedAF-1 (Schwalb et al., 1995, 1996). In addition, in culture, AF-1 wasshown to induce the expression of many of the gene products known to beassociated with axon regeneration in vivo (Petrausch et al., 2000). Asshown here, the mammalian vitreous fluid contains a small molecule withbiophysical properties and bioactivity similar to those of goldfishAF-1. Both AF-1 and the vitreous-derived factor are hydrophilic, eluteas a coherent peak on a gel-filtration column, and induce similar levelsof axon growth from goldfish RGCs in a 6-thioguanine-sensitive andNBTI-insensitive fashion (this study; Schwab et al., 1996; andunpublished observations). Upon further purification, the low molecularweight factor from bovine vitreous was found to contain one componentthat carried most of the biological activity and a second component,which, although not sufficient to induce axon growth by itself, enhancedthe effects of the active factor. Through a combination ofultrafiltration, differential solubility, gel-filtration chromatography,and normal-phase HPLC, we isolated the active component from thevitreous and found by mass spectrometry that it was a carbohydrate withthe formula C₆H₁₂O₆. Testing multiple monosaccharides with this formularevealed a high degree of specificity for stimulating outgrowth. Forgoldfish RGCs, the position of the hydroxyl groups on carbon atoms 3-5is highly constrained, as is stereospecificity, whereas on carbon 2,either the mannose or glucose configuration stimulates growth, as doesthe substitution of an amide group. The fact that low micromolarconcentrations were required to stimulate growth even in the presence ofhigh concentrations of pyruvate and galactose provides one indicationthat the axon-promoting effects of the monosaccharides are independentof energy metabolism. This is further indicated by the dissociationbetween cell survival and axon growth noted throughout the studies.Interestingly, mannose-6- and glucose-6-phospate also stimulated growth,though to a lesser extent than the monosaccharides themselves. Becausethe phosphate derivatives can not get into the cell, it is likely thatthey are acting upon an extracellular sensor. The possibility thatglucose and mannose act extracellularly is further indicated by theobservation that their effect on goldfish RGCs is not diminished by3-O-methyl glucose, an inhibitor of glucose transport. An inhibitor ofhexose kinases did not block the axogenic effects of glucose or mannosedespite diminished cell survival. Together, these findings lend furthersupport to the idea that the effects of glucose and mannose on ougrowthare likely to be through an extracellular sensor and to be independentof energy metabolism or intracellular conversion to another product. Inmammalian RGCs, the dissociation between the axogenic and energeticroles of the carbohydrates is even clearer, since only mannosestimulated outgrowth; glucose at levels up to 5 mM had no axon-promotingeffects.

Cyclic AMP may play several roles in facilitating axon growth. Inneonatal rat RGCs, the ability of trophic factors to stimulate cellsurvival requires cAMP, which in at least some instances enables thecognate receptors to translocate from the cytoplasm to the cell membrane(Meyer-Franke et al., 1995; Shen et al., 1999; Goldberg et al., 2002).Even when the survival of RGCs is made trophic factor-independent byoverexpression of the bcl-2 gene, RGCs still require elevated cAMP to beable to extend axons in response to trophic factor stimulation (Goldberget al., 2002). In growth cones, intracellular cAMP levels have a rapideffect in determining whether various extracellular signals result inattraction or repulsion, and a delayed effect in mediating the “priming”effect of trophic factors for overcoming growth inhibition by myelin orspecific myelin proteins (Cai et al, Neuron, ca. 1999). This lattereffect of cAMP is protein-synthesis dependent, and is related toenhanced expression of Arginase I and its products, polyamines, whichare sufficient to overcome myelin inhibition (Cai et al., 2002). In ourstudies, the role of cAMP is not likely to be related to polyaminesynthesis, since our culture medium already contains high concentrations(100 μM) of putrescine. However, cAMP is required to enable mannose toupregulate expression of GAP-43. In the case of goldfish RGCs, outgrowthwas not enhanced by elevating intracellular cAMP levels.

In summary, our results show that the low molecular weight factor thatstimulates axon outgrowth in goldfish retinal ganglion cells and the lowmolecular weight factor that enhances the response of mature rat RGCs toother growth factors are monosaccharides. Goldfish RGCs show arelatively nonselective growth response to low micromolar concentrationsof either glucose, mannose, or glucosamine, and this does not requireelevation of intracellular cAMP. Thus, the great abundance of thesesugars may help explain the efficient regeneration of the goldfish opticnerve that occurs spontaneously in vivo. In mammals, althoughregenerative failure has been ascribed to inhibitory myelin proteins andto the glial scar at the injury site, these barriers can be overcome toa large extent by intracellular manipulations that cause a macrophageinflux into the eye (Berry et al., 1996; Leon et al., 2000; Fischer etal., 2000, 2001; Yin et al., 2003). Our finding that adult rat RGCsregenerate their axons in response to a macrophage-derived factor in thepresence of mannose and elevated cAMP indicates that monosaccharides mayalso play an important role in axon regeneration in higher vertebrates.

The following Examples 3 and 4 are formulations useful for intraocular,periocular or retrobulbar injection or perfusion.

Example 3

Component % w/v D-mannose 0.1 Dibasic sodium phosphate 0.2 HPMC 0.5Polysorbate 80 0.05 Benzalkonium chloride 0.01 Sodium chloride 0.75Edetate disodium 0.01 NaOH/HCl q.s. to pH 7.4 Purified water q.s. to100% Cremophor EL 10 Tromethamine 0.12 Boric acid 0.3 Mannitol 4.6Edetate disodium 0.1 Benzalkonium chloride 0.1 NaOH/HCl q.s. to pH 7.4Purified water q.s. to 100%

Example 4

Component % w/v Oncomodulin 0.1 D-mannose 0.1 cAMP modulator 0.1 Dibasicsodium phosphate 0.2 HPMC 0.5 Polysorbate 80 0.05 Benzalkonium chloride0.01 Sodium chloride 0.75 Edetate disodium 0.01 NaOH/HCl q.s. to pH 7.4Purified water q.s. to 100% Cremophor EL 10 Tromethamine 0.12 Boric acid0.3 Mannitol 4.6 Edetate disodium 0.1 Benzalkonium chloride 0.1 NaOH/HClq.s. to pH 7.4 Purified water q.s. to 100%Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

REFERENCES

All references cited herein and throughout the specification are herebyincorporated by reference in their entirety.

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1. A method of treating a neurological disorder in a subject in needtherof comprising administering to the subject a therapeuticallyeffective amount of a hexose.
 2. The method of claim 1, wherein saidhexose is selected from the group consisting of D-mannose, gulose andglucose-6-phosphate.
 3. The method of claim 1, further comprisingadministering to the subject a cAMP modulator.
 4. The method of claim 3,wherein said cAMP modulator is non-hydrolyzable cAMP analogues,adenylate cyclase activators, macrophage-derived factors that stimulatecAMP, macrophage activators, calcium ionophores, membranedepolarization, phosphodiesterase inhibitors, specific phosphodiesteraseIV inhibitors, beta2-adrenoreceptor inhibitors or vasoactive intestinalpeptide.
 5. The method of claim 1, further comprising administering tosaid subject a macrophage-derived factor.
 6. The method of claim 5,wherein the macrophage-derived factor is oncomodulin.
 7. The method ofclaim 5, wherein the macrophage-derived factor is TGF-β.
 8. The methodof claim 1, wherein the treatment reverses neuronal damage.
 9. Themethod of claim 1, wherein the treatment alleviates a neurologicaldisorder.
 10. The method of claim 1, wherein the disorder is selectedfrom the group consisting of traumatic brain injury, stroke, cerebralaneurism, spinal cord injury, Parkinson's disease, amyotrophic lateralsclerosis, Alzheimer's disease, diffuse cerebral cortical atrophy,Lewy-body dementia, Pick disease, mesolimbocortical dementia, thalamicdegeneration, Huntington chorea, cortical-striatal-spinal degeneration,cortical-basal ganglionic degeneration, cerebrocerebellar degeneration,familial dementia with spastic paraparesis, polyglucosan body disease,Shy-Drager syndrome, olivopontocerebellar atrophy, progressivesupranuclear palsy, dystonia musculorum deformans, Hallervorden-Spatzdisease, Meige syndrome, familial tremors, Gilles de la Tourettesyndrome, acanthocytic chorea, Friedreich ataxia, Holmes familialcortical cerebellar atrophy, Gerstmann-Straussler-Scheinker disease,progressive spinal muscular atrophy, progressive balbar palsy, primarylateral sclerosis, hereditary muscular atrophy, spastic paraplegia,peroneal muscular atrophy, hypertrophic interstitial polyneuropathy,heredopathia atactica polyneuritiformis, optic neuropathy,ophthalmoplegia, retina or optic nerve damage.
 11. The method of claim1, wherein the hexose is administered by introduction into a region ofneuronal injury.
 12. The method of claim 1, wherein the hexose isintroduced into the cerebrospinal fluid of the subject.
 13. The methodof claim 1, wherein the hexose is introduced to the subjectintrathecally.
 14. The method of claim 1, wherein the hexose isintroduced into a region selected from the group consisting of acerebral ventricle, the lumbar area, and the cistema magna of thesubject.
 15. The method of claim 1, wherein the hexose is administeredtopically to the eye of the subject or by intraocular injection.
 16. Themethod of claim 1, wherein the subject is a mammal.
 17. The method ofclaim 16, wherein the mammal is a human.
 18. The method of claim 1,wherein said neurological disorder is a spinal cord injury.
 19. Themethod of claim 18, wherein the spinal cord injury is characterized bymonoplegia, diplegia, paraplegia, hemiplegia and quadriplegia.
 20. Themethod of claim 10, wherein the damage to the optic nerve is the resultof glaucoma.
 21. The method of claim 10, wherein the damage to theretina is the result of macular degeneration.
 22. An article ofmanufacture comprising packaging material and a pharmaceutical agentcontained within said packaging material, wherein said packagingmaterial comprises a label which indicates said pharmaceutical may beadministered, for a sufficient term at an effective dose, for treating aneurological disorder together with a pharmaceutically acceptablecarrier, wherein the pharmaceutical agent comprises D-mannose.
 23. Thearticle of claim 22, wherein the article further comprises a cAMPmodulator.
 24. The article of claim 22, wherein the article furthercomprises oncomodulin.
 25. A pharmaceutical formulation comprisingD-mannose and a cAMP modulator, and a pharmaceutically acceptablecarrier.
 26. The pharmaceutical formulation of claim 25, furthercomprising oncomodulin.